1. Field of the Invention
The invention relates to screening assays for compounds effecting TRAF-receptor interactions and is useful for the identification of agonists and antagonists of TRAF-receptor interactions.
2. Description of Related Art
Tumor necrosis factor (TNF) receptor superfamily members regulate cellular proliferation, differentiation, and apoptosis in inflammatory and immune responses. Signaling through TNF receptor superfamily members is initiated by oligomerization of the receptors with trimeric ligands bringing intracellular domains in close proximity. Signal transduction through many of these receptors is mediated in part by a recently identified family of proteins termed TNF receptor-associated factors (TRAFs). Six TRAF family members have been identified. Cao, Z., et al (1996) Nature 383, 443-446; Cheng, G. et al 995 Science 267, 1494-1498; Rothe, M., et al (1994) Cell 78, 681-692; Sato, T., et al (1995) FEBS Letters 358, 113-118; Hu, H. M., et al (1994) J. Biol. Chem. 269, 30069-30072; Mosialos, G., et al (1995) Cell 80, 389-399; Regnier, C. H., et al (1995)J. Biol. Chem. 270,25715-25721; Nakano, H., et al (1996) J. Biol. Chem. 271, 14661-14664. Subsets of TRAF proteins have been shown to interact with the TNF receptor family members TNFR2, CD40, CD30, LTBR, ATAR, OX-40, and 4-1BB. Cheng et al (1995); Rothe et al (1994); Sato et al (1995); Hu et al (1994); Nakano et al (1996); Ishida, T. K., et al (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9437-9442; Ishida, T., et al (1996) J. Biol. Chem. 271, 28745-28748; Boucher, L. M., et al (1997) Biochem. Biophys. Res. Commun. 233, 592-600; Lee, S. Y., et al (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9699-9703; Gedrich, R. W., et al (1996) J. Biol. Chem. 271, 12852-12858; Marsters, S. A., et al (1997) J. Biol. Chem. 272, 14029-14032; Aizawa, S. et al (1997) J. Biol. Chem. 272, 2042-2045; Arch, R. H. et al (1998) Molec. Cell Biol. 18, 558-565; Devergne, O., et al (1996) Molec. Cell Biol. 16, 7098-7108; VanArsdale, T. L., et al (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 2460-2465. The conserved C-terminal region of TRAFs (TRAF(NC), also delineated the TRAF domain) binds to receptor cytoplasmic domains. Cheng et al (1995); Takeuchi, M., et al (1996) J. Biol. Chem. 271, 19935-19942; Rothe, M., et al (1995) Science 269, 1424-1427. The initial event in signaling is thought to be mediated by a transient recruitment of TRAF proteins following receptor cross-linking. Kuhne, M. R., et al (1997) J. Exp. Med. 186, 337-342. The interaction of TRAF proteins with cross-linked receptor cytoplasmic domains is therefore a critical step in TNF receptor family member signaling and determines the specificity of the resulting cellular response.
Thus, it is apparent that there is a clear need for a quantitative binding assay for TRAF-receptor interactions and which has the modular flexibility to make possible the introduction of simple modifications in order to measure the interaction of any TNF receptor cytoplasmic domain (or TRAF-binding protein) with any of the six TRAF proteins. Such an assay would be useful for identification of specific agonists or antagonists of these interactions.